γH2AX foci induced by γ rays and 125IdU decay

Purpose: γH2AX foci formation was investigated after γ irradiation and after accumulating ¹²⁵IdU decays to study the DNA double strand break (dsb) damage repair response in human breast cancer cells, MCF‐7.

Materials and methods: Confocal laser scanning microscopy (CLSM) was used to detect γH2AX foci formed in response to DNA dsbs induced by 0, 0.5, 1, 2 and 5?Gy γ irradiation and ¹²⁵IdU decays accumulated at ?90°C in human breast cancer cells, MCF‐7. ¹²⁵IdU treated cells were labeled with 4 different concentrations of ¹²⁵IdU and then accumulated decays for 6, 19 or 35 days. γH2AX foci formation time for all experiments was 1 hour at 37°C. Visual confirmation of γH2AX foci was achieved by digital imaging (histogram analysis or profile analysis) and by standardizing the scored number of foci. The average numbers of γH2AX foci formed per cell after γ irradiation or accumulated ¹²⁵IdU decays were determined by counting red voxels or counting γH2AX foci in propidium iodide (PI) counterstained nuclei by eye in optically sectioned cells.

Results: Control, unirradiated MCF‐7 cells had an average of 1.7 γH2AX foci per cell and an average of 32 γH2AX foci were scored for cells irradiated with 1?Gy γ rays. The data for doses up to approximately 1?Gy was a good linear fit (r²=0.97) indicating that the assay is sensitive to low doses of γ rays. The average number of γH2AX foci scored in control cells that were frozen and thawed but not irradiated (=2.3) was not statistically significantly different from controls that were not frozen and thawed. The average number of γH2AX foci was linearly related (r²=0.98) to low numbers (<200 decays/cell) of ¹²⁵IdU decays indicating that the assay is also sensitive to low numbers of accumulated ¹²⁵IdU decays. At ¹²⁵I decays greater than 200 decays/cell, the average number of γH2AX foci plateaued. Regression analysis of the data for 0–140 ¹²⁵IdU decays per cell was used to calculate the rate of γH2AX foci formation (=0.26 foci per ¹²⁵I decay).

Conclusions: The γH2AX foci formation assay is sensitive to low doses of γ rays and accumulated ¹²⁵I decays. When ¹²⁵IdU decays were accumulated at ?90°C (to overcome confounding DNA damage repair processes that occur during simultaneous ¹²⁵IdU incorporation and decay accumulation at 37°C), 0.26 γH2AX foci were formed per ¹²⁵IdU decay. Methods used to incorporate ¹²⁵I decay may modulate the number of γH2AX foci scored in cells.     

Cytotoxicity of 125I-oestrogen decay in non-oestrogen receptor-expressing human breast cancer cells, MDA-231 and oestrogen receptor-expressing MCF-7 cells

PURPOSE: To compare the cytotoxicity of 125I-oestrogen (E-17alpha[125I]iodovinyl-11betamethoxyoestradiol or 125IVME2) decay accumulation in human breast adenocarcinoma cells that do not express oestrogen receptor (ER) (MDA-231 cells) with human breast adenocarcinoma cells that do express ER (MCF-7 cells). MATERIALS AND METHODS: MDA-231 cells were labelled with 125IVME2 or [125I]iododeoxyuridine (125IdU), frozen for decay accumulation, thawed and then plated for colony formation. gamma-irradiation survival was also determined. A whole-cell 3H-oestrogen-binding assay and a specific-binding assay were used to detect ER. RESULTS: No MDA-231 cell killing by accumulated 125IVME2 decays (up to 440 dpc) was observed but ER-positive MCF-7 cells were killed by 125IVME2 (D(o)=28 dpc). MDA-231 cells were not significantly more radioresistant to gamma-rays (D(o)=1.7Gy for MDA-231 cells; 1 Gy for MCF-7 cells) or to 125IdU decays (D(o)= 44dpc for MDA-231 cells; 30 dpc for MCF-7 cells). No ER were detected in MDA-231 cells. CONCLUSIONS: ER-negative cells, MDA-231, are not killed by 125IVME2 decay accumulation. It is speculated that without ER (required to translocate the 125IVME2 to its nuclear target), formation of the 125IVME2-ER-DNA oestrogen-response element (ERE) complex and subsequent specific irradiation of the DNA at the ERE cannot occur. These results support the hypothesis that the nuclear genome is a critical target for radiation-induced cell death.     

Cytotoxicity of 125 I-oestrogen decay in non-oestrogen receptor-expressing human breast cancer cells,MDA-231 and oestrogen receptor-expressing MCF-7 cells

Purpose : To compare the cytotoxicity of 125 I-oestrogen (E-17 α [ 125 I]iodovinyl-11 β methoxyoestradiol or 125 IVME2) decay accumulation in human breast adenocarcinoma cells that do not express oestrogen receptor (ER) (MDA-231 cells) with human breast adenocarcinoma cells that do express ER (MCF-7 cells). Materials and methods : MDA-231 cells were labelled with 125 IVME2 or [ 125 I]iododeoxyuridine (125 IdU), frozen for decay accumulation, thawed and then plated for colony formation. γ-irradiation survival was also determined. A whole-cell 3 H-oestrogen-binding assay and a specific-binding assay were used to detect ER. Results : No MDA-231 cell killing by accumulated 125 IVME2 decays (up to 440 dpc) was observed but ER-positive MCF-7 cells were killed by 125 IVME2 (D o =28 dpc). MDA-231 cells were not significantly more radioresistant to γ-rays (D o =1.7Gy for MDA-231 cells; 1 Gy for MCF-7 cells) or to 125 IdU decays (D o = 44dpc for MDA-231 cells; 30 dpc for MCF-7 cells). No ER were detected in MDA-231 cells. Conclusions : ER-negative cells, MDA-231, are not killed by 125 IVME2 decay accumulation. It is speculated that without ER (required to translocate the 125 IVME2 to its nuclear target), formation of the 125 IVME2-ER-DNA oestrogen-response element (ERE) complex and subsequent specific irradiation of the DNA at the ERE cannot occur. These results support the hypothesis that the nuclear genome is a critical target for radiation-induced cell death.     

Measurement of DNA double-strand breaks in CHO cells at various stages of the cell cycle using pulsed field gel electrophoresis: calibration by means of 125I decay.

Experiments were performed to calibrate a recently developed pulsed field gel electrophoresis assay, the asymmetric field inversion gel electrophoresis (AFIGE), for the measurement of double-strand breaks (dsb) in the DNA of mammalian cells. Calibration was carried out by means of 125I decay accumulation under conditions preventing repair, and is based on the observation that each 125I decay in the DNA produces approximately one dsb. Iodine was incorporated into DNA in the form of 5'-iododeoxyuridine and decay accumulation was allowed in cells kept frozen at -80 degrees C. Since widely different DNA damage dose-response curves were obtained in cells exposed to X-rays in various phases of the cell cycle, calibration was performed using synchronized populations of cells that were allowed to accumulate DNA damage in G1, G1/S, mid-S, and G2 + M. For this purpose the fraction of activity (in DNA) released from the plug (FAR) was measured and correlated to the number of 125I decays accumulated during the elapsed period of time. Fluctuations in the FAR per 125I decay were observed throughout the cell cycle that were similar to those previously reported for the FAR per Gy of X-rays. Comparison of the FAR per 125I decay with the FAR per Gy gave an induction of 21 +/- 3, 31 +/- 3, 21 +/- 3 and 26 +/- 8 dsb per Gy per diploid DNA complement for G1, G1/S, S, and G2 + M cells, respectively. The results suggest that the observed fluctuations in the FAR per Gy throughout the cycle reflect cell-cycle-associated differences in the physicochemical properties of the DNA molecules that alter their electrophoretic mobility, rather than variations in the induction of dsb per Gy, i.e. the sensitivity of the assay fluctuates throughout the cycle. We propose that similar phenomena underlie the observed fluctuations throughout the cell cycle in the fraction of activity eluted per Gy in the non-unwinding filter elution assay. 125I decays accumulated at 4 degrees C in partly purified DNA from cells embedded in agarose plugs and lysed immediately, gave FAR identical to those obtained with cells kept frozen. This finding suggests that indirect effects do not significantly contribute to DNA damage induction by 125I decay, and indicate that calibration of electrophoresis techniques for dsb measurements can be carried out using this simplified protocol.     

Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X-rays

PURPOSE: Exposure to ionizing radiation results in phosphorylation of histone H2AX (gammaH2AX) at sites of DNA double-strand breaks. To determine the relationship between gammaH2AX formation and radiosensitivity, the rate of formation and loss of gammaH2AX were examined in several cultured cell lines following exposure to 253 kV X-rays. MATERIALS AND METHODS: Flow and image cytometry were both performed using a mouse monoclonal antibody against gammaH2AX. Immunoblotting was used to confirm cell line-dependent differences in antibody staining. Cell lines examined included V79 and CHO-K1 hamster cells, the human tumour cell lines SiHa, WiDr, DU145, WIL-2NS, HT144, HCC1937 and U87, and the normal cell strain HFL1. Radiosensitivity was measured using a standard clonogenic assay. RESULTS: Using flow cytometry, gammaH2AX formation was detected 1 h after doses as low as 20 cGy. Peak levels of gammaH2AX were observed within 15-30 min after irradiation and both the rate of radiation-induced gammaH2AX formation and loss were cell type dependent. Maximum levels of gammaH2AX formation were lower for HT144 cells mutant for the ataxia telangiectasia gene. Half-times of loss after irradiation ranged from 1.6 to 7.2 h and were associated with a decrease in the total number of foci per cell. The half-time of loss of gammaH2AX was correlated with clonogenic survival for 10 cell lines (r2=0.66). CONCLUSIONS: GammaH2AX can be detected with excellent sensitivity using both flow and image analysis. The rate of gammaH2AX loss may be an important factor in the response of cells to ionizing radiation, with more rapid loss and less retention associated with more radioresistant cell lines.     

Computational analysis of the number,area and density of γ-H2AX foci in breast cancer cells exposed to 111In-DTPA-hEGF or γ-rays using Image-J software

Purpose: To develop a simple method for the quantification of γ-H2AX focus number, density and size.

Methods: MDA-MB-468 human breast cancer cells were treated overnight with ¹¹¹In-diethylenetriaminepentaacetic acid human epidermal growth factor (¹¹¹In-DTPA-hEGF, 0–142 kBq/pmol) or exposed to γ-radiation to induce DNA double strand breaks (DSB). DNA DSB formation was evaluated by detection of phosphorylated histone H2AX on serine 139 (γ-H2AX) using immunofluorescence. Confocal microscopy was used to capture images of γ-H2AX foci and cell nuclei. Image-J software with customized macros was used to quantify γ-H2AX foci.

Results: The number of γ-H2AX foci per nucleus scored using Image-J correlated strongly with the number scored using direct visual confirmation (coefficient of determination, R² = 0.950; 60 nuclei scored). The mean density (grayscale values per pixel), area and integrated density (IntDen) of individual foci increased linearly as the specific radioactivity (SR) increased up to 67 kBq/pmol (R² values of 0.826, 0.964, 0.978, respectively). The mean number of foci per nucleus, the combined area of γ-H2AX foci per nucleus and the IntDen per nucleus also increased linearly with SR, giving R² values of 0.926, 0.974 and 0.983, respectively. Similar linear relationships were observed with the γ-ray absorbed dose up to 3.0 Gy.

Conclusions: The density, area and IntDen of individual foci, as well as the number of γ-H2AX foci, total focus area and IntDen per nucleus were successfully quantified using Image-J with customized macros.     

Assessment of DNA damage produced by 125I-triplex-forming oligonucleotides in cells

PURPOSE: Triplex-forming oligodeoxyribonucleotides (TFOs) bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. When labeled with Auger-electron-emitting radioisotopes, TFOs are able to damage the target gene in a process named antigene radiotherapy. We compared radiotoxicity and the amount of DNA damage produced within cultured cells by two 125I-labeled TFOs, one with a single target in the genome and another with multiple targets. MATERIALS AND METHODS: Radiotoxicity was measured by clonogenic assay while DNA damage was assessed by the number of histone gamma-H2AX foci formed at the sites of DNA double strand breaks (DSBs). RESULTS: The TFO with multiple nuclear targets was 1.7 fold more radiotoxic and produced on average 1.9 fold more gamma-H2AX foci per cell than the TFO with a single target. CONCLUSION: Since the two methods gave comparable results, measuring the number of gamma-H2AX foci per decay may be a useful procedure for the assessment of cytotoxic effects and the intranuclear localization of radionuclides when they produce DSBs.     

NATO Advanced Study Institute on ‘Photosensitization: Molecular,Cellular and Medical Aspects’ (Held at the Royal Military College of Canada,Kingston, Canada from 5 to 18 July 1987)

Summary

Experiments were performed to calibrate a recently developed pulsed field gel electrophoresis assay, the asymmetric field inversion gel electrophoresis (AFIGE), for the measurement of double-strand breaks (dsb) in the DNA of mammalian cells. Calibration was carried out by means of ¹²⁵I decay accumulation, under conditions preventing repair, and is based on the observation that each ¹²⁵I decay in the DNA produces approximately one dsb. Iodine was incorporated into DNA in the form of 5′-iododeoxyuridine and decay accumulation was allowed in cells kept frozen at ? 80°C. Since widely different DNA damage dose-response curves were obtained in cells exposed to X-rays in various phases of the cell cycle, calibration was performed using synchronized populations of cells that were allowed to accumulate DNA damage in G₁, G₁/S, mid-S, and G₂ + M. For this purpose the fraction of activity (in DNA) released from the plug (FAR) was measured and correlated to the number of ¹²⁵I decays accumulated during the elapsed period of time. Fluctuations in the FAR per ¹²⁵I decay were observed throughout the cell cycle that were similar to those previously reported for the FAR per Gy of X-rays. Comparison of the FAr per ¹²⁵I decay with the FAR per Gy gave an induction of 21 ± 3, 31 ± 3, 21 ± 3 and 26 ± 8 dsb per Gy per diploid DNA complement for G₁, G₁/S, S, and G₂ + M cells, respectively. The results suggest that the observed fluctuations in the FAR per Gy throughout the cycle reflect cell-cycle-associated differences in the physicochemical properties of the DNA molecules that alter their electrophoretic mobility, rather than variations in the induction of dsb per Gy, i.e. the sensitivity of the assay fluctuates throughout the cycle. We propose that similar phenomena underlie the observed fluctuations throughout the cell cycle in the fraction of activity eluted per Gy in the non-unwinding filter elution assay. ¹²⁵I decays accumulated at 4°C in partly purified DNA from cells embedded in agarose plugs and lysed immediately, gave FAR identical to those obtained with cells kept frozen. This finding suggests that indirect effects do not significantly contribute to DNA damage induction by ¹²⁵I decay, and indicate that calibration of electrophoresis techniques for dsb measurements can be carried out using this simplified protocol.     

Dosimetry and risk from low- versus high-LET radiation of Auger events and the role of nuclide carriers

PURPOSE: To analyse the lethality to mammalian cells of (125)I-decays in DNA, in antipyrine in the whole cell and in oligodeoxynucleotides in the nucleus outside DNA as a function of Auger event-site and number. MATERIALS AND METHODS: Auger events cause both low- and high-linear energy transfer energy depositions including charge neutralization at the daughter nuclide. Microdosimetry allows the expression of absorbed dose to a defined micromass and the number of such events at given sites. Published data were used to relate micromass dose and event number to the dose to reduce survival to 37% of the initial survival (D37). RESULTS: The D37 of (125)I-decays in DNA was 0.1 Gy in terms of absorbed dose to the cell nucleus and about 30 in terms of average decays per nucleus or whole cell. The D37 of (125)I-decays in antipyrine was 1.5 Gy for absorbed dose to the cell nucleus, about 250 in terms of average decays per nucleus and about 2 x 10(3) for average decays per whole cell. (125)I-decays in oligodeoxynucleotides were much less toxic than (125)I-decays in antipyrine by a factor of about 25 in terms of average absorbed dose to the cell nucleus, by a factor or about 40 in terms of average decays per cell nucleus and by a factor of six in terms of average decays per whole cell. CONCLUSION: The unexpected low toxicity of (125)I-decays in nuclear oligodeoxynucleotides outside the DNA in comparison with (125)I-decays in antipyrine in the nucleus or the whole cell demands further attention on the role of oligodeoxynucleotides in altering cellular radiation sensitivity.     

60Coγ射线诱导EJ细胞DNA损伤及γH2AX 表达的研究

目的 探讨不同剂量⁶⁰Coγ射线对EJ细胞DNA损伤的情况,不同剂量⁶⁰Coγ 射线照射EJ细胞后诱导磷酸化组蛋白H2AX焦点形成,以及与γH2AX表达量的关系。方法 单细胞凝胶电泳检测DNA链断裂损伤情况。免疫荧光法检测不同剂量γ 射线照射后立即、以及2 Gy γ 射线照射后不同时间的EJ细胞中γH2AX焦点的数量。流式细胞分析法检测不同剂量γ 射线照射后EJ细胞中γH2AX 蛋白表达量的变化。结果 单细胞凝胶电泳结果显示,γ射线照射后DNA损伤情况明显加重,随照射剂量的增加,细胞尾矩不断加大,0 Gy组尾矩为0.24,4 Gy照射组尾矩为5.26;免疫荧光结果显示,随着照射剂量的增加,γH2AX焦点数目及大小均明显增加,照射的剂量范围从0.1~4 Gy均可检测,且照射剂量和焦点形成数目之间存在剂量-效应关系,0.1 Gy照射组每个细胞中的焦点数平均达12.37个,4 Gy照射组每个细胞中的焦点数平均达46个;2 Gy γ 射线照射后24 h仍可检测到γH2AX焦点,随时间延长焦点数目减少、强度减弱,具有时间依赖性。流式细胞检测结果表明,γ 射线照射后γH2AX 蛋白表达量明显增加,呈现明显量效关系,0.1和4 Gy照射组γH2AX阳性细胞表达率分别为7.4%和29.2%。结论 免疫荧光法检测照射后γH2AX焦点数目比其他实验方法更能敏感、直观的反映DNA损伤及修复情况,有望成为检测辐射损伤的理想生物指标。     

Cytotoxicity of 125I decay in the DNA double strand break repair deficient mutant cell line, xrs-5.

The survival of parental Chinese hamster ovary (CHO) K1 cells and the DNA double strand break (DSB) repair deficient mutant, xrs-5 was determined after accumulation of 125I decays. Both CHO and xrs-5 cells were extremely sensitive to accumulated 125I decays. The D0 values for CHO and xrs-5 cells were 40 and approximately 7 decays per cell, respectively. The difference in cell survival between CHO and xrs-5 cells was not due to differences in overall 125IUdR incorporation, differences in labelling index (LI) or differences in plating efficiency (PE). Relative biological effectiveness (RBE) values calculated relative to 137Cs gamma radiation survival values (D0 and D10) were higher in xrs-5 cells compared with CHO cells. Although both CHO and xrs-5 cells have high RBE values that correspond to a high sensitivity of CHO and xrs-5 cells to 125I decay. The higher RBE observed for xrs-5 cells in combination with the known repair defect in xrs-5 cells support the idea that unrepaired DNA double strand breaks are lethal to the cell.     

Assessment of DNA damage produced by 125I‐triplex‐forming oligonucleotides in cells

Purpose: Triplex‐forming oligodeoxyribonucleotides (TFOs) bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. When labeled with Auger‐electron‐emitting radioisotopes, TFOs are able to damage the target gene in a process named antigene radiotherapy. We compared radiotoxicity and the amount of DNA damage produced within cultured cells by two ¹²⁵I‐labeled TFOs, one with a single target in the genome and another with multiple targets.

Materials and methods: Radiotoxicity was measured by clonogenic assay while DNA damage was assessed by the number of histone γ‐H2AX foci formed at the sites of DNA double strand breaks (DSBs).

Results: The TFO with multiple nuclear targets was 1.7 fold more radiotoxic and produced on average 1.9 fold more γ‐H2AX foci per cell than the TFO with a single target.

Conclusion: Since the two methods gave comparable results, measuring the number of γ‐H2AX foci per decay may be a useful procedure for the assessment of cytotoxic effects and the intranuclear localization of radionuclides when they produce DSBs.     

Relationship between induction of phosphorylated H2AX and survival in breast cancer cells exposed to 111In-DTPA-hEGF

The Auger electron-emitting radiopharmaceutical 111In-diethylenetriaminepentaacetic acid human epidermal growth factor (111In-DTPA-hEGF) binds the epidermal growth factor receptor (EGFR), is internalized, and translocates to the nucleus. The purpose of this study was to investigate the relationship between EGFR expression, DNA damage, and cytotoxicity in cells exposed to 111In-DTPA-hEGF. METHODS: Breast cancer cell lines with a range of EGFR expression levels were exposed to 111In-DTPA-hEGF or gamma-radiation. The cell lines (followed by number of EGFR per cell in parentheses) were MDA-MB-468 (1.3 x 10(6)), MDA-MB-231 (1.3 x 10(5)), and MCF-7 (1.5 x 10(4)). The proportion of radioactivity partitioning into the nucleus was measured by cell fractionation. DNA double-strand breaks were evaluated using the gamma-H2AX assay. Clonogenic survival assays were used to measure cytotoxicity. RESULTS: All data are presented as mean +/- SD. The amount of 111In-DTPA-hEGF that translocated to the nucleus (in mBq/nucleus) in MDA-MB-468, MDA-MB-231, and MCF-7 cells incubated with 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h was 131 +/- 6, 8.1 +/- 0.1, and 1.1 +/- 0.9, respectively. The number of gamma-H2AX foci per nucleus was 35 +/- 15, 19 +/- 10, and 1.7 +/- 0.3, respectively. A reduction in the surviving fraction (SF) in MDA-MB-468 (0.013 +/- 0.001) and MDA-MB-231 (0.5 +/- 0.1) but not in MCF-7 cells after exposure to 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h has been demonstrated. The SF of MDA-MB-468 cells after exposure to DTPA-EGF (43 nM) and 111In-acetate (5.2 MBq/mL) for 20 h was 0.5 +/- 0.1 and 0.53 +/- 0.05, respectively. MDA-MB-468 was the most sensitive of the cell lines to gamma-irradiation, with an SF after 2 Gy of 0.45 +/- 0.04, compared with 0.7 +/- 0.1 and 0.8 +/- 0.1 for MCF-7 and MDA-MB-231, respectively. The number of gamma-H2AX foci per nucleus in MDA-MB-468 cells correlated with the concentration, specific activity, and incubation time of 111In-DTPA-hEGF. CONCLUSION: DNA damage caused by 111In-DTPA-hEGF correlates with the EGFR expression level of the exposed cells and with concentration, specific activity, and incubation time of 111In-DTPA-hEGF. The gamma-H2AX assay may be a useful biomarker to predict and monitor the outcome of treatment with 111In-DTPA-hEGF.     

gamma-H2AX foci formation in peripheral blood lymphocytes of tumor patients after local radiotherapy to different sites of the body: dependence on the dose-distribution, irradiated site and time from start of treatment

PURPOSE: To evaluate the relationship between an estimated integral total body radiation dose delivered and phosphorylated histone H2AX protein (gamma-H2AX) foci formation in peripheral blood lymphocytes of cancer patients. MATERIAL AND METHODS: gamma-H2AX formation was quantified as the mean number of foci per lymphocyte (N(meanH2AX)) and the percentage of lymphocytes with > or =n foci. The integrated total body radiation dose was estimated from the dose volume histogram of patient's body corrected for the proportion of the body scanned by computed tomography for 3D treatment planning. RESULTS: There was a strong linear correlation between the mean number of gamma-H2AX foci per lymphocyte in the peripheral blood sample and integrated total body radiation dose (r = 0.83, p < 0.0001). The slope of the relationship was dependent on the site of body irradiated. In comparison to chest irradiation with a slope of 8.7 +/- 0.8 foci Gy(-1), the slopes for brain, upper leg and pelvic sites were significantly shallower by -4.7, -4.3, and -3.8 Gy(-1), respectively (p < 0.0001), while the slope for upper abdomen irradiation was significantly larger by 9.1 +/- 2.6 Gy(-1) (p = 0.0007). There was a slight time effect since the start of radiotherapy on the slopes of the in vivo dose responses leading to shallower slopes (-1.5 +/- 0.7 Gy(-1), p = 0.03) later (> or =10 day) during radiotherapy. After in vitro irradiation, lymphocytes showed 10.41 +/- 0.12 foci per Gy with no evidence of inter-individual heterogeneity. CONCLUSIONS: gamma-H2AX measurements in peripheral lymphocytes after local radiotherapy allow the estimation of the applied integral body dose. The site and time dependence have to be considered.     

Dosimetry and risk from low‐ versus high‐LET radiation of Auger events and the role of nuclide carriers

Purpose: To analyse the lethality to mammalian cells of ¹²⁵I‐decays in DNA, in antipyrine in the whole cell and in oligodeoxynucleotides in the nucleus outside DNA as a function of Auger event‐site and number.

Materials and methods: Auger events cause both low‐ and high‐linear energy transfer energy depositions including charge neutralization at the daughter nuclide. Microdosimetry allows the expression of absorbed dose to a defined micromass and the number of such events at given sites. Published data were used to relate micromass dose and event number to the dose to reduce survival to 37% of the initial survival (D₃₇).

Results: The D₃₇ of ¹²⁵I‐decays in DNA was 0.1?Gy in terms of absorbed dose to the cell nucleus and about 30 in terms of average decays per nucleus or whole cell. The D₃₇ of ¹²⁵I‐decays in antipyrine was 1.5?Gy for absorbed dose to the cell nucleus, about 250 in terms of average decays per nucleus and about 2×10³ for average decays per whole cell. ¹²⁵I‐decays in oligodeoxynucleotides were much less toxic than ¹²⁵I‐decays in antipyrine by a factor of about 25 in terms of average absorbed dose to the cell nucleus, by a factor or about 40 in terms of average decays per cell nucleus and by a factor of six in terms of average decays per whole cell.

Conclusion: The unexpected low toxicity of ¹²⁵I‐decays in nuclear oligodeoxynucleotides outside the DNA in comparison with ¹²⁵I‐decays in antipyrine in the nucleus or the whole cell demands further attention on the role of oligodeoxynucleotides in altering cellular radiation sensitivity.     

Cell loss from viable and necrotic tumor regions after local gamma irradiation measured by 125I-UdR

Using a tracer technique, loss of cells from perivascular and average tumor cells of the syngeneic mammary adenocarcinoma EO 771 in male C57Bl/6J mice may be measured in the living animal, by the use of 125-labelled 5-iodo-2'-deoxyuridine (125I-UdR). It was the purpose of this paper to compare measurements in vivo with those made in vitro following local 60Co-gamma irradiation in the absorbed dose range from 10 to 27.5 Gy, incorporation of radioactivity into DNA of tumor cells and activity loss from labelled tumor cells were measured externally by a special scintillation counter device. In addition, by injecting the vital dye "light green" into the mice the I-125-activity of the stained viable and unstained necrotic regions were separately measured for loss of activity following gamma irradiation. A comparison was made between radiation induced growth delay and the depression of 125I-UdR incorporation into DNA of the proliferating tumor cells. After local tumor irradiation with a dose of 27.5 Gy 60Co gamma rays an enhancement of the activity loss by 0.5% per hour was externally observed for the perivascular tumor cell population. A lower enhancement of 0.4% per hour was externally registered in the average tumor cell population. Both values were evaluated relative to sham-irradiated control tumors. The measurements on isolated tumors were in comparatively good agreement with the external values. The activity loss rate from the viable, euoxic tissue increased by 0.4% per hour after 27.5 Gy 60Co gamma rays and by 0.3% per hour in the average cell population, the latter representing a mixture of euoxic and hypoxic cells. The results demonstrate, that the external measurements are a good indicator for radiation effects under in vivo-conditions.     

Induction and quantification of gamma-H2AX foci following low and high LET-irradiation

PURPOSE: To investigate quantitatively the induction and rejoining of DNA double strand breaks (DSB) in V79-4 and xrs-5 Chinese hamster cells and HF19 human fibroblast cells, using the phosphorylation of the histone protein H2AX (gamma-H2AX) as an indicator of DSB, exposed to low doses of either low linear energy transfer (LET) (60)Co gamma-rays or high LET a-particles. MATERIALS AND METHODS: Cells were irradiated with low or high LET (20 - 2000 mGy). The gamma-H2AX foci were detected using immunohistochemistry and quantified by image analysis. RESULTS: The number of DSB determined 30 min post gamma-irradiation at 37 degrees C is 12.2 (+/-1.5), 13.5 (+/-1.6) and 19.1 (+/-1.7) foci/cell/Gy for V79-4, xrs-5 and HF19 cells respectively, comparable with levels detected in V79-4 cells using pulse field gel electrophoresis. 6 h post gamma-irradiation, gamma-H2AX foci levels in V79-4 and HF19 cells approach control levels but remain higher in DSB repair deficient xrs-5 cells. Gamma-H2AX foci levels remain significantly higher than controls at 6 h in a-irradiated cells. CONCLUSIONS: Gamma-radiation and alpha-radiation induced the phosphorylation of H2AX in response to DSB at low doses; the variation in the rate of dephosphorylation of induced foci are dependent both on radiation quality and cell characteristics.     

Induction of lethal bystander effects in human breast cancer cell cultures by DNA-incorporated Iodine-125 depends on phenotype

Abstract

Purpose: This study uses a three-dimensional cell culture model to investigate lethal bystander effects in human breast cancer cell cultures (MCF-7, MDA-MB-231) treated with ¹²⁵I-labeled 5-iodo-2 -deoxyuridine (¹²⁵IdU). These breast cancer cell lines respectively form metastatic xenografts in nude mice in an estrogen-dependent and independent manner.

Materials and methods: In the present study, these cells were cultured in loosely-packed three-dimensional architecture in a Cytomatrix? carbon scaffold. Cultures were pulse-labeled for 3 h with ¹²⁵IdU to selectively irradiate a minor fraction of cells, and simultaneously co-pulse-labeled with 0.04 mM 5-ethynyl-2′-deoxyuridine (EdU) to identify the radiolabeled cells using Click-iT^® EdU and flow cytometry. The cultures were then washed and incubated for 48 h. The cells were then harvested, serially diluted, and seeded for colony formation. Aliquots of cells were subjected to flow cytometry to determine the percentage of cells labeled with ¹²⁵IdU/EdU. Additional aliquots were used to determine the mean ¹²⁵I activity per labeled cell. The percentage of labeled cells was about 15% and 10% for MCF-7 and MDA cells, respectively. This created irradiation conditions wherein the cross-dose to unlabeled cells was small relative to the self-dose to labeled cells. The surviving fraction relative to EdU-treated controls was measured.

Results: Survival curves indicated significant lethal bystander effect in MCF-7 cells, however, no significant lethal bystander effect was observed in MDA-MB-231 cells.

Conclusions: These studies demonstrate the capacity of ¹²⁵IdU to induce lethal bystander effects in human breast cancer cells and suggest that the response depends on phenotype.     

Leukocyte DNA damage after multi-detector row CT: a quantitative biomarker of low-level radiation exposure

PURPOSE: To prospectively determine if gammaH2AX (phosphorylated form of H2AX histone variant)-based visualization and quantification of DNA damage induced in peripheral blood mononuclear cells (PBMCs) can be used to estimate the radiation dose received by adult patients who undergo multidetector computed tomography (CT). MATERIALS AND METHODS: After institutional review board approval and written informed patient consent were obtained, eight women and five men (mean age, 63.8 years) who would be undergoing chest-abdominal-pelvic CT or chest CT only were recruited. Venous blood samples obtained before scanning were exposed to different radiation doses in vitro and incubated for 5-30 minutes to obtain reference values of gammaH2AX focus yield. Additional blood samples were taken 5-30 minutes after CT. Leukocytes were isolated, fixed, and stained for gammaH2AX expression. The gammaH2AX focus yields were determined with fluorescence microscopy, and the radiation doses delivered during CT were estimated by comparing post-CT focus yields with in vitro pre-CT focus yields. These CT radiation doses were compared with doses calculated by using phantom dosimetry and Monte Carlo data sets. Data were analyzed by using linear regression, the dispersion index test, and the contaminated Poisson method. RESULTS: Compared with the gammaH2AX focus yields in blood samples taken before CT (0.06 focus per cell+/-0.01 [mean+/-standard error of mean]), the yields in blood samples taken 5 minutes after chest-abdominal-pelvic CT (0.52 focus per cell+/-0.02) were 8-10-fold higher and corresponded to a mean radiation dose of 16.4 mGy (95% confidence interval: 15.1, 17.7). The mean yield of 0.24 focus per cell+/-0.04 in one patient after chest CT corresponded to a mean radiation dose of 6.3 mGy+/-1.4. In comparison, phantom dosimetry-calculated total blood doses were 13.85 mGy with whole-body CT and 5.16 mGy with chest CT. CONCLUSION: gammaH2AX focus yield in blood cells may be a useful quantitative biomarker of human low-level radiation exposure.     

Cytotoxicity of 125I Decay in the DNA Double Strand Break Repair Deficient Mutant Cell Line,Xrs-5

The survival of parental Chinese hamster ovary (CHO) K1 cells and the DNA double strand break (DSB) repair deficient mutant, xrs-5 was determined after accumulation of ¹²⁵I decays. Both CHO and xrs-5 cells were extremely sensitive to accumulated ¹²⁵I decays. The D_o values for CHO and xrs-5 cells were 40 and approximately 7 decays per cell, respectively. The difference in cell survival between CHO and xrs-5 cells was not due to differences in overall ¹²⁵IUdR incorporation, differences in labelling index (LI) or differences in plating efficiency (PE). Relative biological effectiveness (RBE) values calculated relative to ¹³⁷Cs gamma radiation survival values (D_o and D₁₀) were higher in xrs-5 cells compared with CHO cells. Although both CHO and xrs-5 cells have high RBE values that correspond to a high sensitivity of CHO and xrs-5 cells to ¹²⁵I decay. The higher RBE observed for xrs-5 cells in combination with the known repair defect in xrs-5 cells support the idea that unrepaired DNA double strand breaks are lethal to the cell.